The guidance and adhesion protein FLRT2 dimerizes in cis via dual small-X3-small transmembrane motifs.

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

AR cooperates with SMAD4 to maintain skeletal muscle homeostasis.

Androgens and androgen-related molecules exert a plethora of functions across different tissues, mainly through binding to the transcription factor androgen receptor (AR). Despite widespread therapeutic use and misuse of androgens as potent anabolic agents, the molecular mechanisms of this effect on skeletal muscle are currently unknown. Muscle mass in adulthood is mainly regulated by the bone morphogenetic protein (BMP) axis of the transforming growth factor (TGF)-ß pathway via recruitment of mothers against decapentaplegic homolog 4 (SMAD4) protein. Here we show that, upon activation, AR forms a transcriptional complex with SMAD4 to orchestrate a muscle hypertrophy programme by modulating SMAD4 chromatin binding dynamics and enhancing its transactivation activity. We challenged this mechanism of action using spinal and bulbar muscular atrophy (SBMA) as a model of study. This adult-onset neuromuscular disease is caused by a polyglutamine expansion (polyQ) in AR and is characterized by progressive muscle weakness and atrophy secondary to a combination of lower motor neuron degeneration and primary muscle atrophy. Here we found that the presence of an elongated polyQ tract impairs AR cooperativity with SMAD4, leading to an inability to mount an effective anti-atrophy gene expression programme in skeletal muscle in response to denervation. Furthermore, adeno-associated virus, serotype 9 (AAV9)-mediated muscle-restricted delivery of BMP7 is able to rescue the muscle atrophy in SBMA mice, supporting the development of treatments able to fine-tune AR-SMAD4 transcriptional cooperativity as a promising target for SBMA and other conditions associated with muscle loss.

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary ß3-subunit.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated ß-subunits. The ß3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length ß3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric ß3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the ß3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the ß3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the ß3-subunit is not required for this clustering but ß3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the ß3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the ß3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the ß3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the ß3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.

Candida albicans is a fungal pathobiont, able to cause epithelial cell damage and immune activation. These functions have been attributed to its secreted toxin, candidalysin, though the molecular mechanisms are poorly understood. Here, we identify epidermal growth factor receptor (EGFR) as a critical component of candidalysin-triggered immune responses. We find that both C. albicans and candidalysin activate human epithelial EGFR receptors and candidalysin-deficient fungal mutants poorly induce EGFR phosphorylation during murine oropharyngeal candidiasis. Furthermore, inhibition of EGFR impairs candidalysin-triggered MAPK signalling and release of neutrophil activating chemokines in vitro, and diminishes neutrophil recruitment, causing significant mortality in an EGFR-inhibited zebrafish swimbladder model of infection. Investigation into the mechanism of EGFR activation revealed the requirement of matrix metalloproteinases (MMPs), EGFR ligands and calcium. We thus identify a PAMP-independent mechanism of immune stimulation and highlight candidalysin and EGFR signalling components as potential targets for prophylactic and therapeutic intervention of mucosal candidiasis.

The architecture of EGFR's basal complexes reveals autoinhibition mechanisms in dimers and oligomers.

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.

3D visualization of additive occlusion and tunable full-spectrum fluorescence in calcite.

From biomineralization to synthesis, organic additives provide an effective means of controlling crystallization processes. There is growing evidence that these additives are often occluded within the crystal lattice. This promises an elegant means of creating nanocomposites and tuning physical properties. Here we use the incorporation of sulfonated fluorescent dyes to gain new understanding of additive occlusion in calcite (CaCO3), and to link morphological changes to occlusion mechanisms. We demonstrate that these additives are incorporated within specific zones, as defined by the growth conditions, and show how occlusion can govern changes in crystal shape. Fluorescence spectroscopy and lifetime imaging microscopy also show that the dyes experience unique local environments within different zones. Our strategy is then extended to simultaneously incorporate mixtures of dyes, whose fluorescence cascade creates calcite nanoparticles that fluoresce white. This offers a simple strategy for generating biocompatible and stable fluorescent nanoparticles whose output can be tuned as required.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms.

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.

A tale of the epidermal growth factor receptor: The quest for structural resolution on cells.

The challenge of determining the architecture and geometry of oligomers of the epidermal growth factor receptor (EGFR) on the cell surface has been approached using a variety of biochemical and biophysical methods. This review is intended to provide a narrative of how key concepts in the field of EGFR research have evolved over the years, from the origins of the prevalent EGFR signalling dimer hypothesis through to the development and implementation of methods that are now challenging the conventional view. The synergy between X-ray crystallography and cellular fluorescence microscopy has become particularly important, precisely because the results from these two methods diverged and highlighted the complexity of the challenge. We illustrate how developments in super-resolution microscopy are now bridging this gap. Exciting times lie ahead where knowledge of the nature of the complexes can assist with the development of a new generation of anti-cancer drugs.

Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution.

There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5-50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.

Nanometric molecular separation measurements by single molecule photobleaching.

Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.

Determining the geometry of oligomers of the human epidermal growth factor family on cells with 7 nm resolution.

Dimerisation, oligomerisation, and clustering of receptor molecules are important for control of the signalling process. There has been a lack of suitable methods for the study and quantification of these processes in cells. Here we describe a protocol for a method that we have named "fluorophore localisation imaging with photobleaching" (FLImP), which uses single molecule localisation and single-step photobleaching to determine the separation of two fluorophores with a resolution of 7 nm or better. We describe the procedures required for the collection of FLImP data, and point out some of the pitfalls that must be avoided for the collection of high resolution data. We also present recent data obtained using FLImP, showing that the intracellular domain of the Epidermal Growth Factor Receptor is not required in the basal state for the receptor to form ordered inactive oligomers in the plasma membrane.

Structure-function relationships and supramolecular organization of the EGFR (epidermal growth factor receptor) on the cell surface.

Dimerization and higher-order oligomerization are believed to play an important role in the activation of the EGFR (epidermal growth factor receptor). Understanding of the process has been limited by the lack of availability of suitable methods for the measurement in cells of distances in the range 10-100 nm, too short for imaging methods and too long for spectroscopic methods such as FRET. In the present article, we review the current state of our knowledge of EGFR oligomerization, and describe results from a new single-molecule localization method that has allowed the quantitative characterization of the distribution of EGFR-EGFR distances in cells. Recent data suggest the involvement of cortical actin in regulating the formation of EGFR complexes.

Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Multicolour single molecule imaging in cells with near infra-red dyes.

BACKGROUND: The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. METHODOLOGY/PRINCIPAL FINDINGS: A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470-1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.

Investigating extracellular in situ EGFR structure and conformational changes using FRET microscopy.

The crystallographic structures of functional fragments of ErbBs have provided excellent insights into the geometry of growth factor binding and receptor dimerization. By placing together receptor fragments to build structural models of entire receptors, we expect to understand how these enzymes are allosterically regulated; however, several predictions from these models are inconsistent with experimental evidence from cells. The opening of this gap underlines the need to investigate intact ErbBs by combining cellular and structural studies into a full picture.

Optics clustered to output unique solutions: a multi-laser facility for combined single molecule and ensemble microscopy.

Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena.

Automated multidimensional single molecule fluorescence microscopy feature detection and tracking.

Characterisation of multi-protein interactions in cellular networks can be achieved by optical microscopy using multidimensional single molecule fluorescence imaging. Proteins of different species, individually labelled with a single fluorophore, can be imaged as isolated spots (features) of different colour light in different channels, and their diffusive behaviour in cells directly measured through time. Challenges in data analysis have, however, thus far hindered its application in biology. A set of methods for the automated analysis of multidimensional single molecule microscopy data from cells is presented, incorporating Bayesian segmentation-based feature detection, image registration and particle tracking. Single molecules of different colours can be simultaneously detected in noisy, high background data with an arbitrary number of channels, acquired simultaneously or time-multiplexed, and then tracked through time. The resulting traces can be further analysed, for example to detect intensity steps, count discrete intensity levels, measure fluorescence resonance energy transfer (FRET) or changes in polarisation. Examples are shown illustrating the use of the algorithms in investigations of the epidermal growth factor receptor (EGFR) signalling network, a key target for cancer therapeutics, and with simulated data.

Human epidermal growth factor receptor (EGFR) aligned on the plasma membrane adopts key features of Drosophila EGFR asymmetry.

The ability of epidermal growth factor receptor (EGFR) to control cell fate is defined by its affinity for ligand. Current models suggest that ligand-binding heterogeneity arises from negative cooperativity in signaling receptor dimers, for which the asymmetry of the extracellular region of the Drosophila EGFR has recently provided a structural basis. However, no asymmetry is apparent in the isolated extracellular region of the human EGFR. Human EGFR also differs from the Drosophila EGFR in that negative cooperativity is found only in full-length receptors in cells. To gain structural insights into the human EGFR in situ, we developed an approach based on quantitative Förster resonance energy transfer (FRET) imaging, combined with Monte Carlo and molecular dynamics simulations, to probe receptor conformation in epithelial cells. We experimentally demonstrate a high-affinity ligand-binding human EGFR conformation consistent with the extracellular region aligned flat on the plasma membrane. We explored the relevance of this conformation to ligand-binding heterogeneity and found that the asymmetry of this structure shares key features with that of the Drosophila EGFR, suggesting that the structural basis for negative cooperativity is conserved from invertebrates to humans but that in human EGFR the extracellular region asymmetry requires interactions with the plasma membrane.

Focal adhesions are sites of integrin extension.

Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)-based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin alpha5beta1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled alpha5beta1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of beta1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

Self-association of calcium-binding protein S100A4 and metastasis.

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.